Researcher Database

Masashi Shingai
Research Center for Zoonosis Control Laboratory for Biologics Development
Associate Professor

Researcher Profile and Settings


  • Research Center for Zoonosis Control Laboratory for Biologics Development

Job Title

  • Associate Professor


  • PhD(Osaka city university)

J-Global ID

Research Interests

  • antibody   Vaccine   Measles   HIV   Influenza virus   

Research Areas

  • Life sciences / Molecular biology / Immunology
  • Life sciences / Molecular biology / Virology

Research Activities

Published Papers

  • Masashi Shingai, Sarah Welbourn, Jason M. Brenchley, Priyamvada Acharya, Eri Miyagi, Ronald J. Plishka, Alicia Buckler-White, Peter D. Kwong, Yoshiaki Nishimura, Klaus Strebel, Malcolm A. Martin
    PLOS PATHOGENS 11 (5) e1004928  1553-7366 2015/05 [Refereed][Not invited]
    For nearly 20 years, the principal biological function of the HIV-2/SIV Vpx gene has been thought to be required for optimal virus replication in myeloid cells. Mechanistically, this Vpx activity was recently reported to involve the degradation of Sterile Alpha Motif and HD domain-containing protein 1 (SAMHD1) in this cell lineage. Here we show that when macaques were inoculated with either the T cell tropic SIVmac239 or the macrophage tropic SIVmac316 carrying a Vpx point mutation that abrogates the recruitment of DCAF1 and the ensuing degradation of endogenous SAMHD1 in cultured CD4(+) T cells, virus acquisition, progeny virion production in memory CD4(+) T cells during acute infection, and the maintenance of set-point viremia were greatly attenuated. Revertant viruses emerging in two animals exhibited an augmented replication phenotype in memory CD4(+) T lymphocytes both in vitro and in vivo, which was associated with reduced levels of endogenous SAMHD1. These results indicate that a critical role of Vpx in vivo is to promote the degradation of SAMHD1 in memory CD4(+) T lymphocytes, thereby generating high levels of plasma viremia and the induction of immunodeficiency.
  • Joseph R. Francica, Zizhang Sheng, Zhenhai Zhang, Yoshiaki Nishimura, Masashi Shingai, Akshaya Ramesh, Brandon F. Keele, Stephen D. Schmidt, Barbara J. Flynn, Sam Darko, Rebecca M. Lynch, Takuya Yamamoto, Rodrigo Matus-Nicodemos, David Wolinsky, Martha Nason, Nicholas M. Valiante, Padma Malyala, Ennio De Gregorio, Susan W. Barnett, Manmohan Singh, Derek T. O'Hagan, Richard A. Koup, John R. Mascola, Malcolm A. Martin, Thomas B. Kepler, Daniel C. Douek, Lawrence Shapiro, Robert A. Seder
    NATURE COMMUNICATIONS 6 6565  2041-1723 2015/04 [Refereed][Not invited]
    Developing predictive animal models to assess how candidate vaccines and infection influence the ontogenies of Envelope (Env)-specific antibodies is critical for the development of an HIV vaccine. Here we use two nonhuman primate models to compare the roles of antigen persistence, diversity and innate immunity. We perform longitudinal analyses of HIV Env-specific B-cell receptor responses to SHIVAD8 infection and Env protein vaccination with eight different adjuvants. A subset of the SHIVAD8-infected animals with higher viral loads and greater Env diversity show increased neutralization associated with increasing somatic hypermutation (SHM) levels over time. The use of adjuvants results in increased ELISA titres but does not affect the mean SHM levels or CDR H3 lengths. Our study shows how the ontogeny of Env-specific B cells can be tracked, and provides insights into the requirements for developing neutralizing antibodies that should facilitate translation to human vaccine studies.
  • Florian Klein, Lilian Nogueira, Yoshiaki Nishimura, Ganesh Phad, Anthony P. West, Ariel Halper-Stromberg, Joshua A. Horwitz, Anna Gazumyan, Cassie Liu, Thomas R. Eisenreich, Clara Lehmann, Gerd Faetkenheuer, Constance Williams, Masashi Shingai, Malcolm A. Martin, Pamela J. Bjorkman, Michael S. Seaman, Susan Zolla-Pazner, Gunilla B. Karlsson Hedestam, Michel C. Nussenzweig
    JOURNAL OF EXPERIMENTAL MEDICINE 211 (12) 2361 - 2372 0022-1007 2014/11 [Refereed][Not invited]
    Antibody-mediated immunotherapy is effective in humanized mice when combinations of broadly neutralizing antibodies (bNAbs) are used that target nonoverlapping sites on the human immunodeficiency virus type 1 (HIV-1) envelope. In contrast, single bNAbs can control simian-human immunodeficiency virus (SHIV) infection in immune-competent macaques, suggesting that the host immune response might also contribute to the control of viremia. Here, we investigate how the autologous antibody response in intact hosts can contribute to the success of immunotherapy. We find that frequently arising antibodies that normally fail to control HIV-1 infection can synergize with passively administered bNAbs by preventing the emergence of bNAb viral escape variants.
  • Masashi Shingai, Olivia K. Donau, Ronald J. Plishka, Alicia Buckler-White, John R. Mascola, Gary J. Nabel, Martha C. Nason, David Montefiori, Brian Moldt, Pascal Poignard, Ron Diskin, Pamela J. Bjorkman, Michael A. Eckhaus, Florian Klein, Hugo Mouquet, Julio Cesar Cetrulo Lorenzi, Anna Gazumyan, Dennis R. Burton, Michel C. Nussenzweig, Malcolm A. Martin, Yoshiaki Nishimura
    JOURNAL OF EXPERIMENTAL MEDICINE 211 (10) 2061 - 2074 0022-1007 2014/09 [Refereed][Not invited]
    It is widely appreciated that effective human vaccines directed against viral pathogens elicit neutralizing antibodies (NAbs). The passive transfer of anti-HIV-1 NAbs conferring sterilizing immunity to macaques has been used to determine the plasma neutralization titers, which must be present at the time of exposure, to prevent acquisition of SIV/HIV chimeric virus (SHIV) infections. We administered five recently isolated potent and broadly acting anti-HIV neutralizing monoclonal antibodies (mAbs) to rhesus macaques and challenged them intrarectally 24 h later with either of two different R5-tropic SHIVs. By combining the results obtained from 60 challenged animals, we determined that the protective neutralization titer in plasma preventing virus infection in 50% of the exposed monkeys was relatively modest (similar to 1:100) and potentially achievable by vaccination.
  • Takaki H, Honda K, Atarashi K, Kobayashi F, Ebihara T, Oshiumi H, Matsumoto M, Shingai M, Seya T
    Molecular immunology 57 (2) 100 - 110 0161-5890 2014/02 [Refereed][Not invited]
  • Hiromi Takaki, Makoto Takeda, Maino Tahara, Masashi Shingai, Hiroyuki Oshiumi, Misako Matsumoto, Tsukasa Seya
    JOURNAL OF IMMUNOLOGY 191 (9) 4740 - 4747 0022-1767 2013/11 [Refereed][Not invited]
    Infection by measles virus (MV) induces type I IFN via the retinoic acid-inducible gene I/melanoma differentiation-associated gene 5/mitochondrial antiviral signaling protein (MAVS) pathway in human cells. However, the in vivo role of the MAVS pathway in host defense against MV infection remains undetermined. CD150 transgenic (Tg) mice, which express human CD150, an entry receptor for MV, with the disrupting IFNR gene (Ifnar(-/-)), are susceptible to MV and serve as a model for MV infection. In this study, we generated CD150Tg/Mavs(-/-) mice and examined MV permissiveness compared with that in CD150Tg/Ifnar(-/-) mice. MV replicated mostly in the spleen of i.p.-infected CD150Tg/Ifnar(-/-) mice. Strikingly, CD150Tg/Mavs(-/-) mice were not permissive to MV in vivo because of substantial type I IFN induction. MV barely replicated in any other organs tested. When T cells, B cells, and dendritic cells (DCs) isolated from CD150Tg/Mavs(-/-) splenocytes were cultured with MV in vitro, only the DCs produced type I IFN. In vitro infection analysis using CD150Tg/Mavs(-/-) DC subsets revealed that CD4(+) and plasmacytoid DCs, but not CD8 alpha(+) and CD8 alpha(-)CD4(-) double negative DCs, were exclusively involved in type I IFN production in response to MV infection. Because CD150Tg/Mavs(-/-) mice turned permissive to MV by anti-IFNAR Ab, type I IFN produced by CD4(+) DCs and plasmacytoid DCs plays a critical role in antiviral protection for neighboring cells expressing IFNAR. Induction of type I IFN in these DC subsets was abolished by the MyD88 inhibitory peptide. Thus, production of type I IFN occurs via the MyD88-dependent and MAVS-independent signaling pathway during MV infection.
  • Shingai M, Nishimura Y, Klein F, Mouquet H, Donau OK, Plishka R, Buckler-White A, Seaman M, Piatak M Jr, Lifson JD, Dimitrov DS, Nussenzweig MC, Martin MA
    Nature 503 (7475) 277 - 280 0028-0836 2013/11 [Refereed][Not invited]
  • Reza Sadjadpour, Olivia K. Donau, Masashi Shingai, Alicia Buckler-White, Sandra Kao, Klaus Strebel, Yoshiaki Nishimura, Malcolm A. Martin
    JOURNAL OF VIROLOGY 87 (15) 8798 - 8804 0022-538X 2013/08 [Refereed][Not invited]
    Neutralization-resistant simian-human immunodeficiency virus AD8 (SHIVAD8) variants that emerged in an infected macaque elite neutralizer targeting the human immunodeficiency virus type 1 (HIV-1) gp120 N332 glycan acquired substitutions of critical amino acids in the V3 region rather than losing the N332 glycosylation site. One of these resistant variants, carrying the full complement of gp120 V3 changes, was also resistant to the potent anti-HIV-1 monoclonal neutralizing antibodies PGT121 and 10-1074, both of which are also dependent on the presence of the gp120 N332 glycan.
  • Masashi Shingai, Olivia K. Donau, Stephen D. Schmidt, Rajeev Gautam, Ronald J. Plishka, Alicia Buckler-White, Reza Sadjadpour, Wendy R. Lee, Celia C. LaBranche, David C. Montefiori, John R. Mascola, Yoshiaki Nishimura, Malcolm A. Martin
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 109 (48) 19769 - 19774 0027-8424 2012/11 [Refereed][Not invited]
    The induction of broadly reacting neutralizing antibodies has been a major goal of HIV vaccine research. Characterization of a pathogenic CCR5 (R5)-tropic SIV/HIV chimeric virus (SHIV) molecular clone (SHIVAD8-EO) revealed that eight of eight infected animals developed cross-reactive neutralizing antibodies (NAbs) directed against an envelope glycoprotein derived from the heterologous HIV-1(DH12) strain. A panel of plasmas, collected from monkeys inoculated with either molecularly cloned or uncloned SHIVAD8 stocks, exhibited cross-neutralization against multiple tier 1 and tier 2 HIV-1 clade B isolates. One SHIVAD8-infected animal also developed NAbs against clades A and C HIV-1 strains. In this particular infected macaque, the cross-reacting anti-HIV-1 NAbs produced between weeks 7 and 13 were directed against a neutralization-sensitive virus strain, whereas neutralizing activities emerging at weeks 41-51 targeted more neutralization-resistant HIV-1 isolates. These results indicate that the SHIVAD8 macaque model represents a potentially valuable experimental system for investigating B-cell maturation and the induction of cross-reactive NAbs directed against multiple HIV-1 strains.
  • Rajeev Gautam, Yoshiaki Nishimura, Wendy R. Lee, Olivia Donau, Alicia Buckler-White, Masashi Shingai, Reza Sadjadpour, Stephen D. Schmidt, Celia C. LaBranche, Brandon F. Keele, David Montefiori, John R. Mascola, Malcolm A. Martin
    JOURNAL OF VIROLOGY 86 (16) 8516 - 8526 0022-538X 2012/08 [Refereed][Not invited]
    There is an urgent need to develop new pathogenic R5 simian/human immunodeficiency viruses (SHIVs) for the evaluation of candidate anti-HIV vaccines in nonhuman primates. Here, we characterize swarm SHIVAD8 stocks, prepared from three infected rhesus macaques with documented immunodeficiency at the time of euthanasia, for their capacity to establish durable infections in macaques following inoculation by the intravenous (i.v.) or intrarectal (i.r.) route. All three viral stocks (SHIVAD8-CE8J SHIVAD8-CK15) and SHIVAD8-CL98) exhibited robust replication in vivo and caused marked depletion of CD4(+) T cells affecting both memory and naive CD4+ T lymphocyte subsets following administration by either route. Eleven of 22 macaques inoculated with the new SHIVAD8 stocks were euthanized with clinical symptoms of immunodeficiency and evidence of opportunistic infections (Pneumocystis, Candida, and Mycobacterium). A single but unique founder virus, also present in the SHIVAD8-CE8J cm swarm stock, was transmitted to two animals following a single i.r. inoculation of approximately 3 50% animal infectious doses, which is close to the threshold required to establish infection in all exposed animals. Because the three new SHIVAD8 viruses are mucosally transmissible, exhibited tier 2 sensitivity to anti-HIV-1 neutralizing antibodies, deplete CD4(+) T lymphocytes in vivo, and induce AIDS in macaques, they are eminently suitable as challenge viruses in vaccine experiments.
  • Laura M. Walker, Devin Sok, Yoshiaki Nishimura, Olivia Donau, Reza Sadjadpour, Rajeev Gautam, Masashi Shingai, Robert Pejchal, Alejandra Ramos, Melissa D. Simek, Yu Geng, Ian A. Wilson, Pascal Poignard, Malcolm A. Martin, Dennis R. Burton
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 108 (50) 20125 - 20129 0027-8424 2011/12 [Refereed][Not invited]
    It is widely believed that the induction of a broadly neutralizing antibody (bNAb) response will be a critical component of a successful vaccine against HIV. A significant fraction of HIV-infected individuals mount bNAb responses, providing support for the notion that such responses could be elicited through vaccination. Infection of macaques with simian immunodeficiency virus (SIV) or SIV/HIV chimeric virus (SHIV) has been widely used to model aspects of HIV infection, but to date, only limited bNAb responses have been described. Here, we screened plasma from 14 R5-tropic SHIV-infected macaques for broadly neutralizing activity and identified a macaque with highly potent cross-clade plasma NAb response. Longitudinal studies showed that the development of broad and autologous NAb responses occurred coincidentally in this animal. Serum-mapping studies, using pseudovirus point mutants and antigen adsorption assays, indicated that the plasma bNAbs are specific for epitopes that include carbohydrates and are critically dependent on the glycan at position 332 of Env gp120. The results described herein provide insight into the development and evolution of a broad response, suggest that certain bNAb specificities may be more rapidly induced by immunization than others, and provide a potential model for the facile study of the development of bNAb responses.
  • Masashi Shingai, Takeshi Yoshida, Malcolm A. Martin, Klaus Strebel
    JOURNAL OF VIROLOGY 85 (19) 9708 - 9715 0022-538X 2011/10 [Refereed][Not invited]
    Human immunodeficiency virus type 1 (HIV-1) Vpu enhances the release of viral particles from infected cells by targeting BST-2/tetherin, a cellular protein inhibiting virus release. The widely used HIV-1(NL4-3) Vpu functionally inactivates human BST-2 but not murine or monkey BST-2, leading to the notion that Vpu antagonism is species specific. Here we investigated the properties of the CXCR4-tropic simian-human immunodeficiency virus DH12 (SHIV(DH12)) and the CCR5-tropic SHIV(AD8), each of which carries vpu genes derived from different primary HIV-1 isolates. We found that virion release from infected rhesus peripheral blood mononuclear cells was enhanced to various degrees by the Vpu present in both SHIVs. Transfer of the SHIV(DH12) Vpu transmembrane domain to the HIV-1(NL4-3) Vpu conferred antagonizing activity against macaque BST-2. Inactivation of the SHIV(DH12) and SHIV(AD8) vpu genes impaired virus replication in 6 of 8 inoculated rhesus macaques, resulting in lower plasma viral RNA loads, slower losses of CD4(+) T cells, and delayed disease progression. The expanded host range of the SHIV(DH12) Vpu was not due to adaptation during passage in macaques but was an intrinsic property of the parental HIV-1(DH12) Vpu protein. These results demonstrate that the species-specific inhibition of BST-2 by HIV-1(NL4-3) Vpu is not characteristic of all HIV-1 Vpu proteins; some HIV-1 isolates encode a Vpu with a broader host range.
  • Yoshiaki Nishimura, Masashi Shingai, Wendy R. Lee, Reza Sadjadpour, Olivia K. Donau, Ronald Willey, Jason M. Brenchley, Ranjini Iyengar, Alicia Buckler-White, Tatsuhiko Igarashi, Malcolm A. Martin
    JOURNAL OF VIROLOGY 85 (20) 10617 - 10626 0022-538X 2011/10 [Refereed][Not invited]
    Evolution of the env gene in transmitted R5-tropic human immunodeficiency virus type 1 (HIV-1) strains is the most widely accepted mechanism driving coreceptor switching. In some infected individuals, however, a shift in coreceptor utilization can occur as a result of the reemergence of a cotransmitted, but rapidly controlled, X4 virus. The latter possibility was studied by dually infecting rhesus macaques with X4 and R5 chimeric simian simian/human immunodeficiency viruses (SHIVs) and monitoring the replication status of each virus using specific primer pairs. In one of the infected monkeys, both SHIVs were potently suppressed by week 12 postinoculation, but a burst of viremia at week 51 was accompanied by an unrelenting loss of total CD4(+) T cells and the development of clinical disease. PCR analyses of plasma viral RNA indicated an env gene segment containing the V3 region from the inoculated X4 SHIV had been transferred into the genetic background of the input R5 SHIV by intergenomic recombination, creating an X4 virus with novel replicative, serological, and pathogenic properties. These results indicate that the effects of retrovirus recombination in vivo can be functionally profound and may even occur when one of the recombination participants is undetectable in the circulation as cell-free virus.
  • Hiromi Takaki, Yumi Watanabe, Masashi Shingai, Hiroyuki Oshiumi, Misako Matsumoto, Tsukasa Seya
    MOLECULAR IMMUNOLOGY 48 (4) 497 - 504 0161-5890 2011/01 [Refereed][Not invited]
    Laboratory-adapted and vaccine strains of measles virus (MV) induce type I interferon (IFN) in infected cells to a far greater extent than wild-type strains. We investigated the mechanisms for this differential type I IFN production in cells infected with representative MV strains. The overexpression of the wild-type V protein suppressed melanoma differentiation-associated gene 5 (MDA5)-induced IFN-beta promoter activity, while this was not seen in A549 cells expressing CD150 transfected with the V protein of the vaccine strain. The V proteins of the wild-type also suppressed poly I:C-induced IFN regulatory factor 3 (IRF-3) dimerization. The V proteins of the wild-type and vaccine strain did not affect retinoic acid-inducible gene 1 (RIG-I)- or toll-IL-1R homology domain-containing adaptor molecule 1 (TICAM-1)-induced IFN-beta promoter activation. We identified an amino acid substitution of the cysteine residue at position 272 (which is conserved among paramyxoviruses) to an arginine residue in the V protein of the vaccine strain. Only the V protein possessing the 272C residue binds to MDA5. The mutation introduced into the wild-type V protein (C272R) was unable to suppress MDA5-induced IRF-3 nuclear translocation and IFN-beta promoter activation as seen in the V proteins of the vaccine strain, whereas the mutation introduced in the vaccine strain V protein (R272C) was able to inhibit MDA5-induced IRF-3 and IFN-beta promoter activation. The other 6 residues of the vaccine strain V sequence inconsistent with the authentic sequence of the wild-type V protein barely affected the IRF-3 nuclear translocation. These data suggested that the structural difference of vaccine MV V protein hampers MDA5 blockade and acts as a nidus for the spread/amplification of type I IFN induction. Ultimately, measles vaccine strains have two modes of IFN-beta-induction for their attenuation: V protein mutation and production of defective interference (DI) RNA. (C) 2010 Elsevier Ltd. All rights reserved.
  • Yoshiaki Nishimura, Masashi Shingai, Ronald Willey, Reza Sadjadpour, Wendy R. Lee, Charles R. Brown, Jason M. Brenchley, Alicia Buckler-White, Rahel Petros, Michael Eckhaus, Victoria Hoffman, Tatsuhiko Igarashi, Malcolm A. Martin
    JOURNAL OF VIROLOGY 84 (9) 4769 - 4781 0022-538X 2010/05 [Refereed][Not invited]
    A new pathogenic R5-tropic simian/human immunodeficiency virus (SHIV) was generated following serial passaging in rhesus macaques. All 13 animals inoculated with SHIVAD8 passaged lineages experienced marked depletions of CD4(+) T cells. Ten of these infected monkeys became normal progressors (NPs) and had gradual losses of both memory and naive CD4(+) T lymphocytes, generated antiviral CD4(+) and CD8(+) T cell responses, and sustained chronic immune activation while maintaining variable levels of plasma viremia (10(2) to 10(5) RNA copies/ml for up to 3 years postinfection [p.i.]). To date, five NPs developed AIDS associated with opportunistic infections caused by Pneumocystis carinii, Mycobacterium avium, and Campylobacter coli that required euthanasia between weeks 100 and 199 p.i. Three other NPs have experienced marked depletions of circulating CD4(+) T lymphocytes (92 to 154 cells/mu l) following 1 to 2 years of infection. When tested for coreceptor usage, the viruses isolated from four NPs at the time of their euthanasia remained R5 tropic. Three of the 13 SHIVAD8-inoculated macaques experienced a rapid-progressor syndrome characterized by sustained plasma viremia of >1 x 107 RNA copies/ml and rapid irreversible loss of memory CD4(+) T cells that required euthanasia between weeks 19 and 23 postinfection. The sustained viremia, associated depletion of CD4(+) T lymphocytes, and induction of AIDS make the SHIVAD8 lineage of viruses a potentially valuable reagent for vaccine studies.
  • Makoto Kubo, Yoshiaki Nishimura, Masashi Shingai, Wendy Lee, Jason Brenchley, Bernard Lafont, Alicia Buckler-White, Tatsuhiko Igarashi, Malcolm A. Martin
    JOURNAL OF VIROLOGY 83 (14) 7099 - 7108 0022-538X 2009/07 [Refereed][Not invited]
    We investigated whether a 28-day course of potent antiretroviral therapy, initiated at a time point ( 48 h postinoculation) following simian immunodeficiency virus (SIV) inoculation when the acquisition of a viral infection was virtually assured, would sufficiently sensitize the immune system and result in controlled virus replication when treatment was stopped. The administration of tenofovir 48 h after SIV inoculation to six Mamu-A*01-negative rhesus macaques did, in fact, potently suppress virus replication in all of the treated rhesus macaques, but plasma viral RNA rapidly became detectable in all six animals following its cessation. Unexpectedly, the viral set points in the treated monkeys became established at two distinct levels. Three controller macaques had chronic phase virus loads in the range of 1 x 10(3) RNA copies/ml, whereas three noncontroller animals had set points of 2 x 10(5) to 8 x 10(5) RNA copies/ml. All of the noncontroller monkeys died with symptoms of immunodeficiency by week 60 postinfection, whereas two of the three controller animals were alive at week 80. Interestingly, the three controller macaques each carried major histocompatibility complex class I alleles that previously were reported to confer protection against SIV, and two of these animals generated cytotoxic T-lymphocyte escape viral variants during the course of their infections.
  • Masashi Shingai, Masahiro Azuma, Takashi Ebihara, Miwa Sasai, Kenji Funami, Minoru Ayata, Hisashi Ogura, Hiroyuki Tsutsumi, Misako Matsumoto, Tsukasa Seya
    INTERNATIONAL IMMUNOLOGY 20 (9) 1169 - 1180 0953-8178 2008/09 [Refereed][Not invited]
    Monocyte-derived dendritic cells (mDCs) recognize viral RNA extrinsically by Toll-like receptor (TLR)3 on the membrane and intrinsically retinoic acid-inducible on the membrane and intrinsically retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5) in the cytoplasm to induce type I IFNs and mDC maturation. When mDCs were treated with live or UV-irradiated respiratory syncytial virus (RSV), early (similar to 4h) induction of IFN-beta usually occurs in other virus infections was barely observed. Live RSV subsequently replicated to activate the cytoplasmic IFN-inducing pathway leading to robust type I IFN induction. We found that RSV initial attachment to cells blocked polyI:C-mediated IFN-beta induction, and this early IFN-beta-modulating event was abrogated by antibodies against envelope proteins of RSV, demonstrating the presence of a IFN-regulatory mode by early RSV attachment to host cells. By IFN-stimulated response element (ISRE) receptor analysis in HEK293 cells, polyI:C- LPS-mediated ISRE activation was dose dependently inhibited by live and inactive RSV to a similar extent. Of the RSV envelope proteins, simultaneously expressed or exogenously added RSV G or soluble G (sG) proteins inhibited TLR/4-mediated ISRE activation in HEK293 cells. sG proteins expressed in cells did not affect the RIG-1/MDA5 pathway but inhibited the TLR adaptor TRIF/TICAM-1 pathway for ISRE activation. Finally, extrinsically added sG protein suppressed the production of IFN-beta in mDCs. Although the molecular mechanism of this extrinsic functional mode of the RSV G glycoprotein (G protein) remains undetermined, G proteins may neutralize the fusion glycoprotein function that promotes IFN-mediated mDC modulation via TCR4 and may cause insufficient raising cell-mediated immunity against RSV.
  • Takashi Ebihara, Masashi Shingai, Misako Matsumoto, Takaji Wakita, Tsukasa Seya
    HEPATOLOGY 48 (1) 48 - 58 0270-9139 2008/07 [Refereed][Not invited]
    Dendritic cell maturation critically modulates antiviral immune responses, and facilitates viral clearance. Hepatitis C virus (HCV) is characterized by its high predisposition to persistent infection. Here, we examined the immune response of human monocyte-derived dendritic cells (MODCs) to the JFH1 strain of HCV, which can efficiently replicate in cell culture. However, neither HCV RNA replication nor antigen production was detected in MoDCs inoculated with JFH1. None of the indicators of HCV interacting with MoDCs we evaluated were affected, including expression of maturation markers (CD80, 83, 86), cytokines (interleukin-6 and interferon-beta), the mixed lymphocyte reaction, and natural killer (NK) cell cytotoxicity. Strikingly, MoDCs matured by phagocytosing extrinsically-infected vesicles containing HCV-derived double-stranded RNA (dsRNA). When MoDCs were cocultured with HCV-infected apoptotic Huh7.5.1 hepatic cells, there was increased CD86 expression and interleukin-6 and interferon-beta production in MoDCs, which were characterized by the potential to activate NK cells and induce CD4(+) T cells into the T helper 1 type. Lipid raft-dependent phagocytosis of HCV-infected apoptotic vesicles containing dsRNA was indispensable to MoDC maturation. Colocalization of dsRNA with Toll-like receptor 3 (TLR3) in phagosomes suggested the importance of TLR3 signaling in the MoDC response against HCV. Conclusion: The JFH1 strain does not directly stimulate MoDCs to activate T cells and NK cells, but phagocytosing HCV-infected apoptotic cells and their interaction with the TLR3 pathway in MoDCs plays a critical role in MoDC maturation and reciprocal activation of T and NK cells.
  • Megumi Higuchi, Aya Matsuo, Masashi Shingai, Kyoko Shida, Akihiro Ishii, Kenji Funami, Yasuhiko Suzuki, Hiroyuki Oshiumi, Misako Matsumoto, Tsukasa Seya
    DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY 32 (2) 147 - 155 0145-305X 2008 [Refereed][Not invited]
    Human Toll-like receptor 2 (TLR2) subfamily recognizes bacterial lipoproteins (BLP) and peptidoglycan (PGN). According to the genome information, chicken has structural orthologs of TLRs1 and 2, in addition to TLRs3, 4, 5 and 7. Chicken has two additional TLRs, TLR15 and TLR21, whose orthologs human lacks. The chicken (ch)TLR1 and 2 genes are individually duplicated to encode for four different proteins, chTLR1-1, 1-2, 2-1 and 2-2, of the TLR2 subfamily. Here we investigated the functional profile of these TLR2 subfamily proteins of chicken. By NF-kappa B reporter assay using HEK293 cells, we found that chTLR2-1 and chTLR1-2 cooperatively signal the presence of PGN. A combination of chTLR2-1 and chTLR1-2 also most efficiently recognized diacylated BLP, macrophage-activating lipopeptide 2 kDa (Malp-2), while the combination of chTLR2-1 and chTLR1-1 failed to recognize Malp-2. All combinations, however, recognized triacylated BLP, Pamm3. Consistent with these results, human TLR2-stimulating mycobacteria preparations, BCG-cell wall and cell lysate of Mycobacterium avium, induced activation of NF-kappa B in cells expressing chTLR2-1 and 1-2 and to lesser extents, cells with chTLR2-2 and either of chTLR1. Strikingly, expression of either of these alone did not activate the reporter for NF-kappa B. These chTLRs are likely to have the combination functional feature as in the human TLR2 subfamily. Confocal and immunoprecipitation analyses of human cell transfectants showed that they cluster on the cell surface by a physical molecular association, causing all of them to merge and coprecipitate. These results suggest that chTLR2 subfamily members discriminate between their ligands by combinational events. (C) 2007 Elsevier Ltd. All rights reserved.
  • Minoru Ayata, Masashi Shingai, Xiaojun Ning, Misako Matsumoto, Tsukasa Seya, Sanae Otani, Toshiyuki Seto, Shinji Ohgimoto, Hisashi Ogura
    VIRUS RESEARCH 130 (1-2) 260 - 268 0168-1702 2007/12 [Refereed][Not invited]
    Measles virus (MV) is the causative agent of subacute sclerosing panencephalitis (SSPE) and viruses isolated from brains of the patients contain numerous mutations. We have previously demonstrated that the hemagglutinin (H) protein of MV SSPE strains can interact with the signaling lymphocyte activation molecule (SLAM) and an unidentified molecule on Vero cells, but not with CD46, as a receptor. The mechanism by which MV SSPE strains can induce cell-cell fusion in SLAM-negative Vero cells is not understood. We report here on the effect of mutations in the fusion (F) proteins of three MV SSPE strains on syncytium formation. The F proteins of the three SSPE strains were functional and co-expression with H protein from the MV wild-type or SSPE strains in this study induced formation of large syncytia in Vero cells as well as in cell lines expressing SLAM or CD46. Expression of chimeric F proteins of SSPE strains showed that amino acid substitutions in the F protein extracellular as well as cytoplasmic domain contributed to enhanced cell-cell fusion in Vero cells. These findings suggest a common molecular mechanism and a key role of the F protein for syncytium formation in cells expressing an unidentified third receptor for MV. (c) 2007 Elsevier B.V. All rights reserved.
  • Differential type IIFN-Inducing abilities of wild-type versus vaccine strains of measles virus
    Masashi Shingai, Takashi Ebihara, Nasim A. Begum, Atsushi Kato, Toshiki Honma, Kenji Matsumoto, Hirohisa Saito, Hisashi Ogura, Misako Matsumoto, Tsukasa Seya
    JOURNAL OF IMMUNOLOGY 179 (9) 6123 - 6133 0022-1767 2007/11 [Refereed][Not invited]
    Laboratory adapted and vaccine strains of measles virus (MV) induced type I IFN in infected cells. The wild-type strains in contrast induced it to a far lesser extent. We have investigated the mechanism for this differential type I IFN induction in monocyte-derived dendritic cells infected with representative MV strains. Laboratory adapted strains Nagahata and Edmonston infected monocyte-derived dendritic cells and activated IRF-3 followed by IFN-,6 production, while wild-type MS failed to activate IRF-3. The viral IRF-3 activation is induced within 2 h, an early response occurring before protein synthesis. Receptor usage of CD46 or CD150 and nucleocapsid (N) protein variations barely affected the strain-to-strain difference in IFN-inducing abilities. Strikingly, most of the IFN-inducing strains possessed defective interference (DI) RNAs of varying sizes. In addition, an artificially produced DI RNA consisting of stem (the leader and trailer of MV) and loop (the GFP sequence) exhibited potential IFN-inducing ability. In this case, however, cytoplasmic introduction was needed for DI RNA to induce type I IFN in target cells. By genesilencing analysis, DI RNA activated the RIG-IIMDA5-mitochondria antiviral signaling pathway, but not the TLR3-TICAM-1 pathway. DI RNA-containing strains induced IFN-beta mRNA within 2 h while the same recombinant strains with no DI RNA required >12 h postinfection to attain similar levels of IFN-beta mRNA. Thus, the stem-loop structure, rather than full genome replication or specific internal sequences of the MV genome, is required for an early phase of type I IFN induction by MV in host cells.
  • Takashi Ebihara, Hisayo Masuda, Takashi Akazawa, Masashi Shingai, Hideaki Kikuta, Tadashi Ariga, Misako Matsumoto, Tsukasa Seya
    INTERNATIONAL IMMUNOLOGY 19 (10) 1145 - 1155 0953-8178 2007/10 [Refereed][Not invited]
    Monocyte-derived dendritic cells (mDCs) and NK cells are reciprocally activate via cytokines and cell-cell contact. Although seven human NKG2D ligands (NKG2DLs), UL16-binding proteins (ULBP) 1, 2, 3 and 4, retinoic acid early transcript 1G (RAET1G) and MHC class I-related chains A and B, have been reported, the differential distribution and roles of these ligands in the maturation of human mDCs have not been elucidated. In the present study, we produced polyclonal antibodies (pAbs) directed against human ULBP1, 2 and 3. All these ULBPs were detected on human mDCs when probed by the pAbs, although their expression profiles were different. We next investigated what kinds of Toll-like receptor agonists and RNA viruses [influenza virus, human respiratory syncytial virus (RSV), measles virus and hepatitis C virus (HCV)] induced the expression of NKG2DLs on mDCs. ULBP1 was up-regulated on mDCs in response to LPS or infection with RSV. The expression of ULBP2 was induced by LPS and poly I:C, indicating that the TIR-containing adapter molecule-1 (TIR domain-containing adaptor-inducing IFN) pathway is associated with ULBP2 induction. Although infection with HCV did not cause up-regulation of NKG2DLs, other RNA virus infections and poly I:C promoted expression of ULBP2 and RAET1G in an IFN-alpha/beta-independent manner. Finally, the over-expression of ULBP1 and 2 on mDCs facilitated NK cell proliferation and IFN-gamma production through a mDC-NK cell interaction in the presence of IL-2. Hence, the results reflect the important role of NKG2DLs on human mDCs in mDC-mediated NK cell activation.
  • Takashi Akazawa, Masashi Shingai, Mwa Sasai, Takashi Ebihara, Norimitsu Inoue, Misako Matsumoto, Tsukasa Seya
    FEBS LETTERS 581 (18) 3334 - 3340 0014-5793 2007/07 [Refereed][Not invited]
    Myeloid dendritic cells (mDCs) play an important role in the initiation of immune responses to cancer and infectious diseases. Toll-like receptors (TLRs) expressed on mDCs recognize microbial products to elicit signals for mDC maturation, including cytokine production, antigen-presentation and induction of effector cells. TLR agonists work as adjuvants to modulate the function of mDCs. In TLR signaling, MyD88 and TRIF/TICAM-1 are major TLR adaptor molecules, which when overexpressed are able to transduce downstream signals without TLR stimuli. We successfully introduced the adaptors into mouse bone marrowderived mDCs using lentiviral vectors. Introduction of MyD88 into mDCs in vitro led to the production of IL-6 and IL-12p40 while introduction of TICAM-1 stimulated interferon (IFN)-alpha production. Expression of TICAM-1, but not MyD88, in mDCs slightly induced the co-stimulatory molecule CD86, while significant upregulation of CD86 was observed in response to other TLR stimuli. Both MyD88 and TICAM-1 augmented allogeneic mixed lymphocyte reaction (MLR). Ex vivo mouse spleen cells pre-exposed to tumor antigen exhibited antitumor cytotoxicity when incubated with MyD88- or TICAM-1 -expressing mDCs. Using mDC adoptive transfer and a syngeneic mouse tumor implant model, we established an antitumor immunotherapy whereby tumor growth is retarded by adaptor-manipulated mDCs. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
  • Takashi Akazawa, Takashi Ebihara, Manabu Okuno, Yu Okuda, Masashi Shingai, Kunio Tsujimura, Toshitada Takahashi, Masahito Ikawa, Masaru Okabe, Norimitsu Inoue, Miki Okamoto-Tanaka, Hiroyoshi Ishizaki, Jun Miyoshi, Misako Matsumoto, Tsukasa Seya
    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 104 (1) 252 - 257 0027-8424 2007/01 [Refereed][Not invited]
    Myeloid dendritic cells (mDCs) recognize and respond to polyl:C, an analog of dsRNA, by endosomal Toll-like receptor (TLR) 3 and cytoplasmic receptors. Natural killer (NK) cells are activated in vivo by the administration of polyl:C to mice and in vitro are reciprocally activated by mDCs, although the molecular mechanisms are as yet undetermined. Here, we show that the TLR adaptor TICAM-1 (TRIF) participates in mDC-derived antitumor INK activation. In a syngeneic mouse tumor implant model (C57BL/6 vs. B16 melanoma with low H-2 expresser), i.p. administration of polyl:C led to the retardation of tumor growth, an effect relied on by INK activation. This NK-dependent tumor regression did not occur in TICAM-1(-/-) or IFNAR(-/-) mice, whereas a normal NK antitumor response was induced in PKR-/-, MyD88(-/-), IFN-beta(-/-), and wild-type mice. IFNAR was a prerequisite for the induction of IFN-alpha/beta and TLR3. The lack of TICAM-1 did not affect IFN production but resulted in unresponsiveness to IL-12 production, mDC maturation, and polyl:C-mediated NK-antitumor activity. This NK activation required NK-mDC contact but not IL-12 function in in vitro transwell analysis. Implanted tumor growth in IFNAR(-/-) mice was retarded by adoptively transferring polyl:C-treated TICACM-1-positive mDCs but not TICAM-1(-/-) mDCs. Thus, TICAM-1 in mDCs critically facilitated mDC-NK contact and activation of antitumor INK, resulting in the regression of low MHC-expressing tumors.
  • Miwa Sasai, Masashi Shingai, Kenji Funami, Mitsutoshi Yoneyama, Takashi Fujita, Misako Matsumoto, Tsukasa Seya
    JOURNAL OF IMMUNOLOGY 177 (12) 8676 - 8683 0022-1767 2006/12 [Refereed][Not invited]
    TLR3 and the cytoplasmic helicase family proteins (retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5)) serve as dsRNA pattern-recognition receptors. In response to poly(I:C), a representative of dsRNA, and viral infection, they have been shown to activate the transcription factor IFN regulatory factor (IRF)-3, which in turn induces activation of the IFN-beta promoter. RIG-I/MDA5 recognizes dsRNA in the cytoplasm, whereas TLR3 resides in the cell surface membrane or endosomes to engage in extracytoplasmic recognition of dsRNA. Recent reports suggest that TLR3 induces cellular responses in epithelial cells in response to respiratory syncytial virus (RSV). The modus for TLR3 activation by RSV, however, remains unresolved. By small interference RNA gene-silencing technology and human cell transfectants, we have revealed that knockdown of NAK-associated protein 1 (NAP1) leads to the down-regulation of IFN-beta promoter activation > 24 h after poly(I:Q or virus (RSV and vesicular stomatitis virus) treatment. NAP1 is located downstream of the adapter Toll-IL-1R homology domain-containing adapter molecule (TICAM)-1 (Toll/IL-IR domain-containing adapter-inducing IFN-beta) in the TLR3 pathway, but TICAM-1 and TLR3 did not participate in the IRF-3 and IFN-beta promoter activation by RSV infection. Virus-mediated activation of the IFN-beta promoter was largely abrogated by the gene silencing of IFN-beta promoter stimulator-1 (mitochondria antiviral signaling (MAVS), VISA, Cardif), the adapter of the RIG-I/MDA5 dsRNA-recognition proteins. In both the TLR and virus-mediated IFN-inducing pathways, I kappa B kinase-related kinase epsilon and TANK-binding kinase I participated in IFN-beta induction. Thus, RSV as well as other viruses induces replication-mediated activation of the IFN-beta promoter, which is intracellularly initiated by the RIG-I/MDA5 but not the TLR3 pathway. Both the cytoplasmic and TLR3-mediated dsRNA recognition pathways converge upon NAP1 for the activation of the IRF-3 and IFN-beta promoter.
  • Wild-type measles virus infection in human CD46/CD150-transgenic mice: CD11c-positive dendritic cells establish systemic viral infection.
    Shingai M, Inoue N, Okuno T, Okabe M, Akazawa T, Miyamoto Y, Ayata M, Honda K, Kurita-Taniguchi M, Matsumoto M, Ogura H, Taniguchi T, Seya T
    Journal of immunology (Baltimore, Md. : 1950) 175 (5) 3252 - 3261 0022-1767 2005/09 [Refereed][Not invited]
  • [Immunologic tests: Membrane cofactor protein (MCP, CD46)].
    Shingai M, Seya T
    Nihon rinsho. Japanese journal of clinical medicine 63 Suppl 7 95 - 97 0047-1852 2005/07 [Refereed][Not invited]
  • Dendritic cell maturation induced by muramyl dipeptide (MDP) derivatives: monoacylated MDP confers TLR2/TLR4 activation.
    Uehori J, Fukase K, Akazawa T, Uematsu S, Akira S, Funami K, Shingai M, Matsumoto M, Azuma I, Toyoshima K, Kusumoto S, Seya T
    Journal of immunology (Baltimore, Md. : 1950) 174 (11) 7096 - 7103 0022-1767 2005/06 [Refereed][Not invited]
  • Seya T, Shingai M, Matsumoto M
    Uirusu 54 (1) 1 - 8 0042-6857 2004/06 [Refereed][Not invited]
  • H Ishida, M Ayata, M Shingai, Matsunaga, I, Y Seto, Y Katayama, N Iritani, T Seya, Y Yanagi, O Matsuoka, T Yamano, H Ogura
    MICROBIOLOGY AND IMMUNOLOGY 48 (4) 277 - 287 0385-5600 2004 [Refereed][Not invited]
    Measles virus is the causative agent of subacute sclerosing panencephalitis (SSPE). The viruses isolated from brain cells of patients with SSPE (called SSPE viruses) are defective in cell-free virus production in vitro. To investigate the cell tropism of three strains of SSPE virus (Osaka-1, Osaka-2, Osaka-3), SSPE virus-infected cell cultures were treated with cytochalasin D to prepare virus-like particles (CD-VLPs). All CD-VLPs formed syncytia after infection in CHO cells expressing CD150 but not in those expressing CD46. In addition, an antibody to CD46 did not block the infection of Vero cells by SSPE CD-VLPs. The results were consistent with our previous suggestion that one or more unidentified receptors might be involved in the entry process. Infection with the CD-VLPs from three SSPE strains was further examined in different human cell lines, including those of neural origin, and was found to induce syncytia in epithelial cells (HeLa and 293T) as well as neuroblastoma cells (IMR-32 and SK-N-SH) with varying efficiency. SSPE CD-VLPs also infected glioblastoma cells (A172) and astrocytoma cells (U-251) but syncytial formation was rarely induced. These epithelial and neural cell lines were not permissive for the replication of wild-type MV. Together with our previous observations, these results suggest that the cell entry receptor is the major factor determining the cell tropism of SSPE viruses. Further studies are necessary to identify other viral and/or cellular factors that might be involved in the replication of SSPE virus in specific neural cells and in the brain.
  • Subcellular localization of Toll-like receptor 3 in human dendritic cells.
    Matsumoto M, Funami K, Tanabe M, Oshiumi H, Shingai M, Seto Y, Yamamoto A, Seya T
    Journal of immunology (Baltimore, Md. : 1950) 171 (6) 3154 - 3162 0022-1767 2003/09 [Refereed][Not invited]
  • M Shingai, M Ayata, H Ishida, Matsunaga, I, Y Katayama, T Seya, H Tatsuo, Y Yanagi, H Ogura
    JOURNAL OF GENERAL VIROLOGY 84 (Pt 8) 2133 - 2143 0022-1317 2003/08 [Refereed][Not invited]
    The vaccine or Vero cell-adapted strains of measles virus (MV) have been reported to use CD46 as a cell entry receptor, while lymphotropic MVs preferentially use the signalling lymphocyte activation molecule (SLAM or CD150). In contrast to the virus obtained from patients with acute measles, little is known about the receptor that is used by defective variants of MV isolated from patients with subacute sclerosing panencephalitis (SSPE), The receptor-binding properties of SSPE strains of MV were analysed using vesicular stomatitis virus pseudotypes expressing the envelope glycoproteins; of SSPE strains of MV. Such pseudotype viruses could use SLAM but not CD46 for entry. The pseudotype viruses with SSPE envelope glycoproteins could enter Vero cells, which do not express SLAM. In addition, their entry was not blocked by the monoclonal antibody to CD46, pointing to another entry receptor for SSPE strains on Vero cells. Furthermore, the unknown receptor(s), distinct from SLAM and CD46, may be present on cell lines derived from lymphoid and neural cells. Biochemical characterization of the receptor present on Vero cells and SK-N-SH neuroblastoma cells was consistent with a glycoprotein. Identification of additional entry receptors for MV will provide new insights into the mechanism of spread of MV in the central nervous system and possible reasons for differences between MVs isolated from patients with acute measles and SSPE.
  • M Ayata, K Komase, M Shingai, Matsunaga, I, Y Katayama, H Ogura
    JOURNAL OF VIROLOGY 76 (24) 13062 - 13068 0022-538X 2002/12 [Refereed][Not invited]
    Numerous mutations are found in subacute sclerosing panencephalitis (SSPE) viruses, and the M gene is the gene most commonly affected. In some SSPE viruses, such as the MF, Osaka-1, Osaka-2, and Yamagata-1 strains, translation of the M protein is complicated by a transcriptional defect that leads to an almost exclusive synthesis of dicistronic P-M mRNA. To understand the molecular mechanisms of this defect, we sequenced the P gene at the P-M gene junction for several virus strains and probed the involvement of several mutations in the readthrough region via their expression in measles virus minigenomes containing different sequences of the P-M gene junction and flanking reporter genes. The deletion of a single U residue in the U tract of the Osaka-1 strain (3'-UAAUAUUUUU-5') compared with the consensus sequence resulted in a marked reduction of the expression of the downstream reporter gene. In addition, the expression of the downstream gene was markedly decreased by (i) the substitution of a C residue in the U tract of the P gene end of the OSA-2/Fr/B strain of the Osaka-2 virus (3'-UGAUAUUCUU-5' compared with the sequence 3'-UGAUAUUUUU-5' from a sibling virus of the same strain, OSA-2/Fr/V), and (ii) the substitution of a G in the sequence of the P gene end of the Yamagata-1 strain at a variable site immediately upstream from the six-U tract (3'-UGAUGUUUUUU-5' instead of 3'-UGAUUUUUUUU-5'). Mutations at the P gene end can account for the readthrough transcription variation at the P-M gene junction, which directly affects M protein expression.
  • Ning, X, M Ayata, M Kimura, K Komase, K Furukawa, T Seto, N Ito, M Shingai, Matsunaga, I, T Yamano, H Ogura
    VIRUS RESEARCH 86 (1-2) 123 - 131 0168-1702 2002/06 [Refereed][Not invited]
    We determined the nucleotide sequence of the fusion (F) gene of three strains (Osaka-1, -2, and -3) of nonproductive variants of measles virus (MV). These viral strains were isolated in Osaka, Japan. from brain tissues of patients with subacute sclerosing panencephalitis (SSPE). Phylogenetic analysis revealed a close relationship among the three strains of SSPE virus. The cytoplasmic tail of the F protein, predicted from sequence analysis of the gene, is altered in all three SSPE strains when compared to the MV field strains. However, the extent and mode of alteration are different in each strain. The F protein of the Osaka-1 strain has six nonconservative amino acid substitutions and a 29-residue elongation of its cytoplasmic tail. The F protein of the Osaka-3 strain has two nonconservative substitutions and a 5-residue truncation of its C-terminus. Although the termination codon is not altered in the F protein of the Osaka-2 strain. five or six amino acids are changed in the cytoplasmic tail of the F protein of the two sibling viruses of this strain. The significance of the altered cytoplasmic domain of the SSPE viruses in the SSPE pathogenesis is discussed. (C) 2002 Elsevier Science B.V. All rights reserved.
  • N Ito, M Ayata, M Shingai, K Furukawa, T Seto, Matsunaga, I, M Muraoka, H Ogura
    JOURNAL OF NEUROVIROLOGY 8 (1) 6 - 13 1355-0284 2002/02 [Refereed][Not invited]
    Two sibling viruses, Fr/V and Fr/B, of the subacute sclerosing panencephalitis (SSPE) virus Osaka-2 strain were isolated from a small biopsy specimen of the brain of an SSPE patient by cocultivation with two different cell lines, Vero and B95a cells, respectively. These two sibling viruses differ from each other in their molecular mechanisms of defective M protein expression. In this study, we found that the Fr/B virus could scarcely form syncytium foci on Vero cells, although the Fr/V virus could do so on both Vero and B95a cells, showing a similar relation of cell tropism between recent field isolates and laboratory strains of the measles virus. Severe neurovirulence of both F/V and Fr/B viruses was observed in hamsters inoculated intracerebrally with less than 100 PFU, in contrast to the negative neurological and pathological findings in hamsters inoculated even with more than 101 PFU of their possible progenitor measles virus. Comparative sequence analysis of inoculated viruses and reisolated viruses from diseased hamster brains showed few variations at a region containing the P-NI gene junction, indicating that the inoculated viruses propagated in the brains and induced neurovirulence. All these results suggest that SSPE virus isolated with a lymphoid cell line is similar in neuropathogenicity to that isolated with a nonlymphoid cell lines, irrespective of differences in the molecular mechanism of M protein defectiveness.

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